Figure 3

The effect of PI3K and MEK1/2 inhibition on RV-induced apoptosis. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). Cells were harvested and analyzed for markers of apoptosis. A – At indicated time points, cell lysates were collected and incubated with artificial caspase substrate Ac-DEVD-pNA. Free pNA due to caspase cleavage was measured at an absorbance of 405 nm. Data represent mean ± S.E. from three experiments, *P < 0.05. B – The number of measurable dead floating cells in the cell culture supernatant was determined by trypan blue exclusion staining at indicated time points. Data represent mean ± S.E. from three experiments, *P < 0.05. C – Total DNA was extracted from detached and monolayer cells at 72 hours p.i. and apoptotic DNA fragments were resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized using UV transillumination. Molecular size markers were run in the left hand lane. D – Light microscopy photographs of cell monolayers at 72 hours p.i., at a magnification of 20X.